t
*£>-•<
; V"
iif.trt C" total energy consumed
P« production, both somatic and
reproductive
R» energy lost through hea'
production (respiration)
U* energy lost o* excreta
F « energy Lost as feces
C-F-A-P4-R+U Or
P (Scope for Growth)-A -IR + U)
FIELD VERIFICATION PROGRAM
(AQUATIC DISPOSAL)
TECHNICAL REPORT D-85-6
UTILITY OF THE SCOPE FOR GROWTH INDEX
TO ASSESS THE PHYSIOLOGICAL IMPACT OF
BLACK ROCK HARBOR SUSPENDED SEDIMENT
ON THE BLUE MUSSEL, MYTILUS EDULIS:
A LABORATORY EVALUATION
by
William G. Nelson, Dianne Black, Donald Phelps
Environmental Research Laboratory
US Environmental Protection Agency
Narragansett, Rhode Island 02882
September 1985
Final Report
Approved For Public Release; Distribution Unlimited
Prepared for DEPARTMENT OF THE ARMY
US Army Corps of Engineers
Washington, DC 20314-1000
and US Environmental Protection Agency
Washington, DC 20460
Monitored by Environmental Laboratory
US Army Engineer Waterways Experiment Station
PO Box 631, Vicksburg, Mississippi 39180-0631
-------
Destroy this report when no longer needed. Do not return
it to the originator.
The findings in this report are not to be construed as an official
Department of the Army position unless so designated
by other authorized documents.
The contents of this report are not to be used for
advertising, publication, or promotional purposes.
Citation of trade names does not constitute an
official endorsement or approval of the use of
such commercial products.
The D-series of reports includes publications of the
Environmental Effects of Dredging Programs:
Dredging Operations Technical Support
Long-Term Effects of Dredging Operations
Interagency Field Verification of Methodologies for
Evaluating Dredged Material Disposal Alternatives
(Field Verification Program)
-------
SUBJECT: Transmittal of Field Verification Program Technical Report Entitled
"Utility of the Scope for Growth Index to Assess the Physiological
Impact of Black Rock Harbor Suspended Sediment on the Blue Mussel,
Mytilys edulis: A Laboratory Evaluation"
TO: All Report Recipients
1. This is one in a series of scientific reports documenting the findings of
studies conducted under the Interagency Field Verification of Testing and
Predictive Methodologies for Dredged Material Disposal Alternatives (referred
to as the Field Verification Program or FVP). This program is a comprehensive
evaluation of environmental effects of dredged material disposal under condi-
tions of upland and aquatic disposal and wetland creation.
2. The FVP originated out of the mutual need of both the Corps of Engineers
(Corps) and the Environmental Protection Agency (EPA) to continually improve
the technical basis for carrying out their shared regulatory missions. The
program is an expansion of studies proposed by EPA to the US Army Engineer
Division, New England (NED), in support of its regulatory and dredging mis-
sions related to dredged material disposal into Long Island Sound. Discus-
sions among the Corps1 Waterways Experiment Station (WES), NED, and the EPA
Environmental Research Laboratory (ERLN) in Narragansett, RI, made it clear
that a dredging project at Black Rock Harbor in Bridgeport, CT, presented a
unique opportunity for simultaneous evaluation of aquatic disposal, upland
disposal, and wetland creation using the same dredged material. Evaluations
were to be based on technology existing within the two agencies or developed
during the six-year life of the program.
3. The program is generic in nature and will provide techniques and inter-
pretive approaches applicable to evaluation of many dredging and disposal
operations. Consequently, while the studies will provide detailed site-
specific information on disposal of material dredged from Black Rock Harbor,
they will also have great national significance for the Corps and EPA.
4. The FVP is designed to meet both Agencies' needs to document the effects
of disposal under various conditions, provide verification of the predictive
accuracy of evaluative techniques now in use, and provide a basis for deter-
mining the degree to which biological response is correlated with bioaccumula-
tion of key contaminants in the species under study. The latter is an
important aid in interpreting potential biological consequences of bioaccumu-
lation. The program also meets EPA mission needs by providing an opportunity
to document the application of a generic predictive hazard-assessment research
strategy applicable to all wastes disposed in the aquatic environment. There-
fore, the ERLN initiated exposure-assessment studies at the aquatic disposal
site. The Corps-sponsored studies on environmental consequences of aquatic
disposal will provide the effects assessment necessary to complement the EPA-
sponsored exposure assessment, thereby allowing ERLN to develop and apply a
hazard-assessment strategy. While not part of the Corps-funded FVP, the EPA
exposure assessment studies will complement the Corps' work, and together the
Corps and the EPA studies will satisfy the needs of both agencies.
-------
SUBJECT: Transmittal of Field Verification Program Technical Report Entitled
"Utility of the Scope for Growth Index to Assess the Physiological
Impact of Black Rock Harbor Suspended Sediment on the Blue Mussel,
Mytilus edulis; A Laboratory Evaluation"
5. In recognition of the potential national significance, the Office, Chief
of Engineers, approved and funded the studies in January 1982. The work is
managed through the Environmental Laboratory's Environmental Effects of
Dredging Programs at WES. Studies of the effects of upland disposal and
wetland creation are being conducted by WES and studies of aquatic disposal
are being carried out by the ERLN, applying techniques worked out at the
laboratory for evaluating sublethal effects of contaminants on aquatic organ-
isms. These studies are funded by the Corps while salary, support facilities,
etc., are provided by EPA. The EPA funding to support the exposure-assessment
studies followed in 1983; the exposure-assessment studies are managed and
conducted by ERLN.
6. The Corps and EPA are pleased at the opportunity to conduct cooperative
research and believe that the value in practical implementation and improve-
ment of environmental regulations of dredged material disposal will be con-
siderable. The studies conducted under this program are scientific in nature
and will be published in the scientific literature as appropriate and in a
series of Corps technical reports. The EPA will publish findings of the
exposure-assessment studies in the scientific literature and in EPA report
series. The FVP will provide the scientific basis upon which regulatory
recommendations will be made and upon which changes in regulatory implementa-
tion, and perhaps regulations themselves, will be based. However, the docu-
ments produced by the program do not in themselves constitute regulatory
guidance from either agency. Regulatory guidance will be provided under
separate authority after appropriate technical and administrative assessment
of the overall findings of the entire program.
Choromokos, Jr., Ph.D., P.E.
Director, Research and Development
U. S. Army Corps of Engineers
Bernard D. Goldstein, H.D.
Assistant Administrator for
Research and Development
U. S. Environmental Protection
Agency
-------
Unclassified
SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered)
REPORT DOCUMENTATION PAGE
1. REPORT NUMBER 2. GOVT ACCESSION NO.
Technical Report D-85-6
4. TITLE (md Subtitle)
UTILITY OF THE SCOPE FOR GROWTH INDEX TO ASSESS
THE PHYSIOLOGICAL IMPACT OF BLACK ROCK HARBOR
SUSPENDED SEDIMENT ON THE BLUE MUSSEL, MYTILUS
EDULIS: A LABORATORY EVALUATION
7. AUTHORO)
William G. Nelson, Dianne Black, Donald Phelps
9. PERFORMING ORGANIZATION NAME AND ADDRESS
US Environmental Protection Agency
Environmental Research Laboratory
Narragansett, Rhode Island 02882
11. CONTROLLING OFFICE NAME AND ADDRESS
DEPARTMENT OF THE ARMY, US Army Corps of Engineers
Washington, DC 20314-1000 and US Environmental
Protection Agency, Washington, DC 20460
14. MONITORING AGENCY NAME A ADDRESSf/f different tram Controlling Olllce)
US Army Engineer Waterways Experiment Station
Environmental Laboratory
PO Box 631, Vicksburg, Mississippi 39180-0631
READ INSTRUCTIONS
BEFORE COMPLETING FORM
3. RECIPIENT'S CATALOG NUMBER
5. TYPE OF REPORT 4 PERIOD COVERED
Final report
6. PERFORMING ORG. REPORT NUMBER
8. CONTRACT OR GRANT NUMBERf.)
10. PROGRAM ELEMENT, PROJECT, TASK
AREA & WORK UNIT NUMBERS
Field Verification Program
(Aquatic Disposal)
12. REPORT DATE
September 1985
13. NUMBER OF PAGES
56
IS. SECURITY CLASS, (ol thl* report)
Unclassified
15a. DECLASSIFI CATION/DOWNGRADING
SCHEDULE
IS. DISTRIBUTION STATEMENT (ol thl. Report)
Approved for public release; distribution unlimited.
17. DISTRIBUTION STATEMENT (ol th* fbetract entered In Block 30, It dltterent (torn Report)
IB. SUPPLEMENTARY NOTES
Available from National Technical Information Service, 5285 Port Royal Road,
Springfield, Virginia 22161.
19. KEY WORDS (Continue on reran* tld» If necoieery and Identity by block number)
Mussels—Environmental aspects (LC) Mytilus edulis (WES)
Dredging—Environmental aspects (LC) Dredging—Connecticut—
Dredged material (WES) . Black Rock Harbor (LC)
Marine pollution (LC)
20. ABSTRACT (Vaatlaue en nrerme eMt H meMMfjr and Identity by block number;
The sensitivity, variability, and reproducibility of the scope for growth
index (SFG) as an indicator of physiological condition was evaluated utilizing
the blue mussel, Mytilus edulis, after exposure to highly contaminated dredged
material. A preliminary experiment was completed to determine a no-observable-
effects-concentration due to suspended reference sediment (REF) alone (50 mg/Jl)
The effect of contaminated dredged material from Black Rock Harbor (BRH) was
then tested using three treatments of suspended sediment: (a) 50 mg/X, of BRH
sediment (100 BRH), (b) 25 mg/£ each of BRH and REF sediment (Continued)
DO/0""
JAM 73
1473
EDITION OF I MOV 65 IS OBSOLETE
Unclassified
SECURITY CLASSIFICATION Of THIS PAGE (Wrten Dele Entered)
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Unclassified
SECURITY CLASSIFICATION OF THIS PAOE(TWl«n Dmtm Bntfnd)
20. ABSTRACT (Continued).
(50-50 BRH/REF), and (c) 50 mg/£ REF sediment alone (100 REF). This 26-day
bioassay demonstrated a significant SFG reduction in mussels exposed to 100 BRH
sediment (-3.63 J/hr) and the 50-50 BRH/REF treatment (-2.32 J/hr) compared to
mussels exposed to 100 REF sediment (2.53 J/hr). This experiment was replicated
to evaluate the reproducibility of the technique. The second experiment produc-
ed similar results with the 100 REF treatment mussels having a significantly
higher SFG (10.22 J/hr) than both the 50-50 BRH/REF (0.51 J/hr) and 100 BRH
(-1.07 J/hr) mussels. The data indicate that in a laboratory exposure the use
of the SFG index with M. edulis provides a sensitive and reproducible technique
for determining the chronic negative impact due to this dredged material.
This investigation is the first phase in developing field-verified bio-
assessment evaluations for the Corps of Engineers and the U.S. Environmental
Protection Agency regulatory program for dredged material disposal. This
report is not suitable for regulatory purposes; however, appropriate assessment
methodologies that are field verified will be available at the conclusion of
this program.
Unclassified
SECURITY CLASSIFICATION OF THIS PAGEflTh.n DM* Entered)
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PREFACE
This report describes work performed by the U.S. Environmental
Protection Agency (EPA), Environmental Research Laboratory,
Narragansett, R.I. (ERLN), as part of the Interagency Field Verifica-
tion of Testing and Predictive Methodologies for Dredged Material
Disposal Alternatives Program (Field Verification Program (FVP)).
The program is sponsored by the Office, Chief of Engineers (OCE),
and administered by the U.S. Army Engineer Waterways Experiment
Station (WES), Vicksburg, Miss., under the purview of the Environmen-
tal Laboratory (EL). The OCE Technical Monitors were Dr. John Hall
and Dr. William L. Klesch. The objective of this interagency
program is to evaluate the environmental consequences of dredged
material disposal under aquatic, wetland, and upland conditions.
The aquatic portion of the FVP study is being conducted by ERLN,
with the wetland and upland portions conducted by WES.
The principal investigators for this aquatic study were Mr.
William Nelson, Ms. Dianne Black, and Dr. Donald Phelps, all of ERLN.
Assistance in the design and maintenance of the laboratory system was
provided by Ms. Melissa Hughes and Mr. Greg Tracey. Laboratory-
cultured algae were also provided by Mr. Tracey. Technical support for
the scope for growth measurements was provided by Mr. William Giles.
In addition, assistance in statistical analysis was provided by Drs.
James Heltshe and Clifford Katz.
The EPA Technical Director for the FVP was Dr. John H. Gentile;
Technical Coordinator was Mr. Walter Galloway; and the Project Manager
was Mr. Allan Beck.
1
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The study was conducted under the direct management of
Dr. Thomas M. Dillon and Dr. Richard K. Peddicord of the Contam-
inant Mobility and Criteria Group (CMCG), Ecosystem Research and
Simulation Division (ERSD), EL; and the general management of
Dr. Charles R. Lee, Chief, CMCG, Mr. Donald L. Robey, Chief, ERSD,
and Dr. John Harrison, Chief, EL. Mr. Charles C. Calhoun, Jr., and
Dr. Robert M. Engler were Program Mangers of the EL Environmental
Effects of Dredging Programs.
During the preparation of this report, COL Tilford C. Creel,
CE, and COL Robert C. Lee, CE, were Commanders and Directors of
WES and Mr. F. R. Brown was Technical Director. At the time of
publication, COL Allen F. Grum, USA, was Director and Dr. Robert W.
Whalin was Technical Director.
This report should be cited as follows:
Nelson, W.G., Black, D., and Phelps, D. 1985. "The Utility
of the Scope for Growth Index to Assess the Physiological
Impact of Black Rock Harbor Suspended Sediment on the Blue
Mussel, Mytilus edulis; A Laboratory Evaluation," Technical
Report D-85-6, prepared by US Environmental Protection
Agency, Narragansett, R.I., for the US Army Engineer Water-
ways Experiment Station, Vicksburg, Miss.
-------
CONTENTS
PREFACE 1
LIST OF FIGURES A
LIST OF TABLES 5
PART I: INTRODUCTION 6
Background. 6
Purpose. 6
Scope 7
PART II: MATERIALS AND METHODS „ 10
Overview. 10
Sediment Collection and Storage 10
Mussel Collection 12
Experimental Design. - 12
Scope for Growth Methods 17
PART III: RESULTS 24
Exposure System Monitoring 24
Physiological Parameters 25
Scope for Growth Index 28
PART IV: DISCUSSION 33
PART V: CONCLUSIONS AND RECOMMENDATIONS 38
REFERENCES 40
APPENDIX A: No-Observable-Effeet-Concentration Test... Al
Introduction A2
Materials and Methods A2
Results A4
Discussion. A9
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LIST OF FIGURES
No. Page
1 Central Long Island Sound disposal site and
South reference site ., 11
2 Black Rock Harbor, Connecticut, source of dredged
material 11
3 Sediment dosing system with chilled water bath
and argon gas supply 15
4 Exposure system used in Black Rock Harbor
experiments. 15
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LIST OF TABLES
No. Page
1 Daily monitoring data for Black Rock Harbor Experiments
One and Two 24
2 The mean (SE) weight-specific clearance rates of mussels
from the two BRH experiments 26
3 The mean (SE) absorption efficiencies of mussels exposed
to three exposure treatments in the two BRH experiments. 26
4 Mean (SE) weight-specific respiration rates of mussels
exposed to three types of suspended sediments 27
5 The mean (SE) ammonia excretion rates for mussels from
the two BRH exposure experiments 28
6 The mean (SE) weight-specific scope for growth values
for the three exposure treatments in the two BRH
experiments 29
7 The mean 0:N ratios of each treatment in Experiments
One and Two 30
8 The coefficients of variation (percent) for each of the
physiological parameters, the SFG index, and the 0:N
ratio 31
9 Mean dry weight and length of mussels from each
treatment in the BRH experiments 32
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UTILITY OF THE SCOPE FOR GROWTH INDEX TO ASSESS THE PHYSIOLOGICAL
IMPACT OF BLACK ROCK HARBOR SUSPENDED SEDIMENT ON THE BLUE MUSSEL,
MYTILUS EDULIS: A LABORATORY EVALUATION
PART I: INTRODUCTION
Background
1. The U.S. Army Corps of Engineers (CE) and the U.S.
Environmental Protection Agency (EPA) are jointly conducting a com-
prehensive Field Verification Program (FVP). The approach being
used in the FVP Is to evaluate and field validate assessment method-
ologies for predicting the environmental impacts of dredged material
disposal in aquatic, upland, and wetland environments. The research,
evaluation, and field verification of the upland and wetland disposal
options will be conducted by the Environmental Laboratory, U.S. Army
Engineer Waterways Experiment Station (WES), Vicksburg, Miss. The
application and field verification of predictive methodologies for
the aquatic disposal option will be conducted by the EPA Environmental
Research Laboratory (ERLN), Narragansett, R.I.
Purpose
2. There are three major objectives in the aquatic portion
of the FVP at ERLN with respect to the scope for growth (SFG) index.
The first objective is to evaluate the sensitivity, variability, and
reproducibility of the SFG index. The blue mussel, Mytilus edulis,
will be exposed to the same level of Black Rock Harbor (BRH) material in
two separate laboratory experiments. The mussels will then be physiolog-
ically assessed using the SFG index, and the results of each experiment
-------
evaluated to establish the accuracy and reproducibility of the tech-
nique. This constitutes the Laboratory Documentation Phase of the
FVP and is the subject of this report.
3. Subsequently, a second objective will reproduce field level
exposures in the laboratory and observe whether laboratory results
accurately predict what is observed in the field. This is termed the
Field Verification Phase. A third objective will determine the degree
of correlation of tissue residues resulting from the bioaccumulation
of contaminants from dredged material and ecologically significant
alterations in organism viability as observed in both the laboratory
and the field.
Scope
4. The SF6 index (Warren and Davis 1967) is a measure of the
energy available to an organism for production, both somatic and
reproductive, after routine metabolic costs are accounted for. This
index has had extensive application with M«_ edulis ranging from the
investigation of the effects of ration levels on mussels (Thompson
and Bayne 1974) to the effects of estuarine pollution levels on
mussels (Widdows, Phelps, and Galloway 1981). In addition, an eco-
logical relevance of the SFG index has been described by Bayne, Clark,
and Moore (1981). They state that a sustained reduction in SFG
results in decreased growth efficiencies, subsequently smaller indi-
viduals, and ultimately reduced fecundity and fitness.
5. While SFG has been proven useful in other applications,
its use in this component of the FVP is to document the usefulness
-------
and reproducibility of the scope for growth index (SFG) as a physiolog-
ical endpoint in Mytilus edulis for measuring chronic effects of highly
contaminated dredged material. In order to fulfill the Laboratory Docu-
mentation requirements of the FVP, the sensitivity of this technique was
tested using a contaminated sediment (BRH sediment) and a reference sedi-
ment as the exposure materials. Reproducibility was assessed by com-
parison of the results from two separate experimental exposures.
6. A very important point must be emphasized at the begining
of this report. SFG may be used to test two different hypotheses.
The first hypothesis is that differences in exposure conditions (i.e.,
levels of suspended particulates, types of sediments, etc.) have no
direct and immediate effect on the SFG index. To test this hypothesis
the conditions under which SFG is measured replicate the experimental
exposure conditions.
7. The second hypothesis is that there is no chronic effect due
to differences between experimental exposures. To test this hypothesis
the conditions under which SFG is measured are standardized and in no
way attempt to replicate the actual experimental exposure conditions.
Because standardized conditions are employed, only relative differences
between the treatments of an experiment can be compared to evaluate
chronic effects. Comparisons of absolute SFG values between experi-
ments are not completely valid and must be made .carefully. It is
this second application that is being evaluated in the present FVP
testing. All statements and comparisons concerning SFG effects and
reproducibility must be considered with this fact in mind.
8
-------
8. This report details two experiments. The first experiment
establishes the effect of a 50-mg/Jl suspended sediment exposure
of: (a) 100 percent BRH sediment (100 BRH), (b) 100 percent reference
sediment (100 REF), and (c) a 50 percent-50 percent mix of each
sediment (50-50 BRH/REF) on the SFG of M^ edulis. The second exper-
iment, a replicate of the first, documents the reproducibility of
the results obtained. In addition, a preliminary experiment (Appendix
A) was completed to establish a "no-observable-effect-concentration"
(NOEC) of suspended reference sediment with respect to the SFG index.
The laboratory exposure levels were selected strictly as the NOEC.
There was no expectation that these levels were in any way represent-
ative of suspended sedimentary levels actually occurring in central
Long Island Sound.
-------
PART II: MATERIALS AND METHODS
Overview
9. The tests described below generally follow methods pre-
scribed in "Standard Practice for Conducting Acute Toxicity Tests
with Fishes, Macroinvertebrates, and Amphibians" (ASTM 1980). Although
the ASTM test methods were not specifically designed for sediment
tests, they provide guidelines for experimental designs, water quality
parameters, statistical analyses, and animal care, handling, and
acclimation.
Sediment Collection and Storage
10. Two sediment types were used to conduct the suspended
particulate tests in this study. The reference sediment (REF) was
collected from the South reference site in Long Island Sound (4l°7.95"N
and 72C52.7"W) by a Sraith-Maclntyre grab (0.2 m ), press sieved through
a 2-mm sieve, and stored at 4°C until used (Figure 1). Black Rock
Harbor (BRH) sediment was collected from the highly contaminated and
industrallzed dredge site (41°9"N and 73°13"W) with a gravity box corer
2
(0.1 m ) to a depth of 1.21 m, thoroughly mixed, press sieved through a
2-mm sieve, and refrigerated (4°C) until used (Figure 2). In all
experiments, sediments were allowed to reach test temperature and mixed
prior to use.
10
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SOUTH REFERENCE
• SITE
Figure 1. Central Long Island Sound disposal site and
South reference site.
MAINTENANCE
DREDGING
Figure 2.
Black Rock Harbor, Connecticut, source of dredged material,
11
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Mussel Collection
11. Mussels for each experiment, the preliminary one (NOEC)
and the two BRH exposures (experiments one and two-, were collected
in a similar manner with a scallop dredge from an uncontaminated
site near Dutch Island in the west passage of Narragansett Bay
(71°24.0'W by 41°29.4'N) from depths ranging between 5 and 10 m.
Collection information for each experiment is listed below:
Collection Experiment Field Field
Experiment Date Begun Temperature °C Salinity °/oo
NOEC 10/7/83
Experiment 1 11/10/83
Experiment 2 3/8/84
10/11/83
11/16/83
3/19/84
17.5
13.0
5.0
31.0
31.0
29.0
The animals were sorted to obtain a size range of 50 to 55 mm shell
length and held in a laboratory flow-through system at ambient
temperature and in unfiltered seawater until the experiment was
initiated. All experiments were run at 15°C. Mussels collected
from the field when the temperature was below 15°C were acclimated
in running unfiltered seawater at a rate of 1°C per day until 15°C
was reached.
Experimental Design
No-observable-effeet-concentration experiment
12. In order to obtain an effect with BRH material it was
believed that the mussels should be exposed to the highest reasonable
level of suspended particulates. The determination of a reasonable
12
-------
particulate level was termed the no-observable-effeet-concentration
(NOEC) experiment. This experiment is detailed in Appendix A.
Briefly, two criteria were used in selecting the NOEC: (a) maximum
exposure to the. suspended sediment, and (b) no significant reduction
in SFG. The approach taken in the NOEC experiment was to expose
mussels to different particulate concentrations (0, 6.25, 12.5, 25,
50, and 100 mg/£) of suspended reference sediment. The results
indicated that mussels in the 50-mg/£ treatment produced pseudofeces
throughout the 28-day experiment, while those in the lower treatment
levels did not. This would maximize processing of, and thus exposure
to, the suspended material through the gastrointestinal tract of
the animal. In addition, mussels from this treatment also exhibited
the highest SFG values. For these reasons, a NOEC level of 50 mg/A
was selected.
Experiments one and two
13. The first experiment in the laboratory documentation
phase of the FVP was designed to establish the sensitivity of the
SFG index as a measure of impact, in this case due to possible effects
of BRH dredged material on M^ edulis. This experiment was repeated
a second time to further document the observed sensitivity and thus
determine the degree of reproducibility. The following methodology
applies to both BRH experiments.
13
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Exposure system
14. Implementation of the experimental design required the
construction of two identical sediment dosing systems to simultaneously
provide either BRH or REF material as suspended sediment. The dosing
system (Figure 3) consisted of conical-shaped slurry reservoirs placed
in a chilled fiberglass chamber, a diaphram pump, a 4-A separatory
funnel, and several return loops that directed the particulate slurry
through the dosing valves. The slurry reservoirs (40 cm diam. x 55 cm
high) contained 40 H of slurry composed of 37.7 A of filtered seawater
and 2.3 A of either BRH or REF sediment. The fiberglass chamber (94 cm
x 61 cm x 79 cm high) was maintained between 4° and 10°C using an
externally chilled water source. (The slurry was chilled to minimize
microbial degradation during the test.) Polypropylene pipes (3.8 cm
diam.) placed at the bottom of the reservoir cones were connected to
the diaphragm pumps (16 to 40 i/min capacity) that had Teflon
diaphragms. These pumps were used to circulate the slurry but minimize
abrasion so that the physical properties and particle sizes of the
material remained as unchanged as possible.
14
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SEPARATORY
FUNNEL
DELIVERY
MANIFOLD
DOSING
VALVE
TO EXPOSURE
SYSTEM
SLURRY
RESERVOIR
Figure 3. Sediment dosing system
with chilled water bath
and argon gas supply.
reference slurry
L
MUSSEL EXPOSURE SYSTEM
seowoler
BRH slurry
mixing
chamber
magnetic/
stirrer
distribution
chamber —
siphons
lo drain
n
leawater
overflow to water both, then to drain
exposure chamber
lo drain
magnetic slirrer
Figure 4. Exposure system used in the Black Rock Harbor
experiments.
15
-------
15. The separatory funnel was connected to the pump and
returned to the reservoir by polypropylene pipes. The separatory
funnel served two functions: (a) to ensure that a .constant head
pressure was provided by the overflow, and (b) to serve as a connec-
tion for the manifold located 4 cm below the constant head level.
The manifold served to distribute the slurry by directing a portion
of the flow from the funnel (through 6 mm Inside diam. polypropylene
tubes) through the Teflon dosing valves (Figure 3) and back to the
reservoir. At the dosing valves, the slurry was mixed with seawater
to provide the desired concentrations for the toxicity tests. Argon
gas was provided at the rate of 200 ml/min to the reservoir and the
separatory funnel to minimize oxidation of the sediment/seawater
slurry. Narragansett Bay seawater filtered (to 15 ym) through
sand filters was used for these experiments. The dosing valves were
controlled by a microprocessor which was programmed to deliver a
pulse with a duration of 0.1 sec up to continuous pulse delivery and
at intervals from once every second to once every hour.
16. The exposure system is shown in Figure 4. In these
experiments the REF and BRH mixing and distribution chambers (Figure 4)
were maintained at 50 mg/£. Exposure conditions were obtained by
siphoning suspended sediment from the appropriate distribution chambers
to produce a combined flow of 300 ml/min in each exposure chamber. The
amount of suspended particulates both entering the exposure chambers
(actual incoming concentration) and the concentration surrounding the
mussels (actual surrounding concentration) were measured daily using a
16
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spectrophotometer. Prior to the experiment, the relationship between
absorbance and dry weight of suspended participates had been determined
by collecting triplicate samples of suspended sediment directly from
the dlluter or by preparing dilutions from the highest concentration.
The dry weight of these samples was measured using the methods reported
in Lake et al. (1984) and their absorbance was measured on the
spectrophotometer. Linear regression analysis of the data established
the relationship between absorbance and dry weight. Analysis of
variance and multiple comparison tests were performed on the suspended
particulate data collected daily during the experiment.
17. The three exposure treatments consisted of an incoming
concentration of 50 mg/& of: (a) 100 percent BRH sediment (100 BRH),
(b) 100 percent REF sediment (100 REF), and (c) a 50 percent-50 percent
mixture of each sediment (50-50 BRH/REF). Forty mussels were exposed
in each treatment and fed Isochrysis aff. galbana (T-Iso) at a rate of
94 mg/mussel/day. On day 26, ten mussels from each treatment were
sampled for SFG measurements. The remaining mussels were distributed
for other end-point determinations. The first experiment was
terminated after 26 days because mortality began to occur in the 100
BRH treatment. Experiment two was stopped after 26 days to replicate
Experiment one.
Scope for Growth Methods
18. Calculation of the SFG index for M^ edulis required the
measurement of four parameters: clearance rate, respiration rate, food
absorption efficiency, and ammonia excretion rate. Because the null
17
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hypothesis being tested was that there would be no chronic effect due
to exposure to BRH material, the SFG measurements were completed under
standardized conditions. All SFG measurements for a given treatment
were completed in the order shown below within 28 hrs after termination
of the experiment and all measurements were performed at 15°C.
Clearance rate
19. Clearance rate is defined as the volume of water completely
cleared of particles >3 microns (ym) in some unit time (Widdows et al.
1979). In the present experiment this was measured by placing mussels
into individual chambers through which 1 ym filtered seawater flowed at
a rate of 75 ml/min. The unicellular algae, T-Iso, was added to the
filtered seawater to deliver an incoming cell concentration of approx-
imately 25,000 cells/ml (about 0.5 mg/&) to each chamber. Each
chamber was gently aerated to ensure that complete mixing and no
settling of algae occurred. Mussels were allowed to acclimate in the
chambers for at least 1 hr prior to any measurements. The incoming
and outgoing particle concentrations for each chamber were then
measured with a Coulter Counter (Model TAII) and substituted into
the following formula to determine clearance rate:
Clearance rate - [(Cl - C2)/C2] X F (1)
where
Cl and C2 - incoming and outgoing particle
concentrations, respectively
F = flow rate in liters/hour through the
chamber.
18
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Respiration rate
20. Respiration rates were measured by isolating each mussel in a
glass respirometer vessel fitted with an electrode designed to measure
the partial pressure of oxygen (P02). The electrode was connected to a
Radiometer oxygen meter (Model PHM71) which was in turn connected to a
strip chart recorder. Each mussel was allowed to acclimate for about
10 minutes in the vessel prior to respiration measurements. This short
acclimation period was found to be adequate by measuring the
respiration rate of several mussels for 1 hr from time of initial
placement into the vessel. There was no change in rate after the first
5 minutes. Seawater containing algae was pumped into the vessel during
this acclimation period at a rate of 80 ml/min to ensure that food was
present in the chamber and that routine metabolic rate was measured.
After acclimation the flow of seawater was stopped and the decline in
P02 was recorded on the strip chart recorder for approximately 30 min.
The respiration rates were calculated using the following formula:
MMHG RESVOL - MUSVOL 60
ML02HR- X SAT02 X X (2)
160 1000 02TIME
where
ML02HR « oxygen consumed per hour by the mussel, ml
MMHG - change in partial pressure of 02 over time, mm mercury
SAT02 - oxygen saturation level of seawater at that
temperature, ml/A
RESVOL - respiration vessel volume, ml
MUSVOL - volume of the mussel, ml
02TIME « time period of the measurement, min
19
-------
Absorption efficiency
21. After completion of the respiration rate measurements, all
fecal material was removed from each feeding chamber. This ensured
that only the algae consumed during the SFG procedures were used in the
absorption efficiency measurements. At the food concentration used in
the SFG measurements, approximately 0.5 mg/&, no pseudofecal production
occurred. The mussels were allowed to feed overnight in the chambers.
Fecal pellets were collected from each chamber with a Pasteur pipette
and filtered onto a 1 ym Nuclepore polycarbonate filter. The filter
was removed to a watch glass where a few drops of isotonic ammonium
formate were added to facilitate removal of the fecal pellets. The
fecal pellets were scraped off with a plastic spatula, deposited onto
small aluminum pans (1 cm square), and placed in a drying oven at 100°C
for 24 hr. Pellets and pans were weighed using a Perkln Elmer
autobalance (Model AD-2Z). Pellets were ashed at 500°C for 4 hr, and
reweighed to determine the ash-free dry weight:dry weight ratio for
the feces. A similar procedure was completed with the cultured algae
to obtain the ash-free dry weighttdry weight ratio of the food.
Absorption efficiencies were calculated for each mussel according to
the method of Conover (1966) using the following formula:
F - E
Absorption Efficiency = X 100 (3)
(1-E) X F
where
F = ash-free dry weighttdry weight ratio of the food
E » ash-free dry weight:dry weight ratio of the feces
20
-------
22. This technique allowed thb calculation of an absorption
efficiency for each mussel. In the past, at this laboratory and in
the literature, fecal material was collected on pre-ashed glass
fiber filters which weighed a great deal more than the dried and
ashed fecal material. The great differential between the weight of
the glass fiber filter and the weight of the dried and ashed fecal
material appeared to introduce an artifact into the data. The sub-
stitution of the lightweight aluminum pans resulted in a 50 percent
reduction in fecal weight variability and the subsequent absorption
efficiency differences between individual mussels.
Ammonia excretion rate
23. Mussels were placed individually into HCl-stripped beakers
containing 300 ml of 1 ym filtered seawater for a period of 3 hr.
Mussels were then removed and a 0.45-ym filtered, 50-ml sample was
collected from each beaker, deposited into acid-stripped polyethylene
bottles, and stored in a freezer at -20°C until analyzed. Ammonia
analyses were completed in duplicate for each sample according to the
method of Bower and Holm-Hansen (1980).
Scope for growth calculations
24. After completion of the physiological measurements, the
length and volume of each mussel were measured and the tissue excised,
dried for 24 hr at 100°C, and weighed. The clearance rates, respira-
tion rates, and ammonia excretion rates were standardized to a 1 g
animal by converting the rates and dry weights to log 10, and fitting
21
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the data to the allometric equation to obtain the fitted parameters, a
and b, as described by Bayne et al. (1981). The weight-specific rates
for each mussel were determined as follows:
BUte
Weight-Specific Rate = (4)
b
Weight
where b = slope in the allometric equations calculated above.
Absorption efficiencies were found to be independent of size (slope=0)
over this narrow size range so absolute values were used.
25. The weight specific values for each mussel were then used to
calculate the SF6 of each individual by substitution into the following
equation:
Scope for Growth = (C X A) - (R + E) (5)
where
C = energy consumed (clearance rate X surrounding food
concentration X energy of food)
A - absorption efficiency
R and E = energy lost through respiration and nitrogen excretion,
respectively
The following energy conversions were used to calculate SFG:
One mg of T-Iso = 4.5 X 107
cells (this experiment)
One mg of T-Iso = 19.24 J (this experiment)
One ml 02 respired - 20.08 J (Crisp 1971)
One mg NH4-N = 24.56 J (Elliot and Davidson 1975)
The energy content of T-Iso was determined by filtering a volume of the
algae onto preweighed glass fiber filters, drying them at 100°C for 24
hr, and reweighing them to determine algal dry weight. They were then
22
-------
analyzed using the dichromate wet oxidation method of Maciolek (1962)
to determine oxygen consumed and the resultant energy content.
26. Another index, the oxygen:nitrogen (0:N) ratio (the atomic
ratio of oxygen consumed to ammonia-nitrogen excreted) can also be
derived from the data obtained for the SFG calculations. This index
was calculated for comparison with the SFG index.
Statistical analysis
27. Differences in physiological data and the resultant SFG
values between each treatment in the NOEC and BRH exposure experiments
were tested using one-way analysis of variance (Snedecor and Cochran
1978). All statistical tests were completed at the 0.05 level of
significance. Tukey's studentized range test was applied to determine
between-treatment differences. Comparison of results between
laboratory experiments were completed using the Student's t-test, also
at the 0.05 significance level. This distinction was made because
laboratory exposure experiments were completed at different points in
time.
23
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PART III: RESULTS
Exposure System Monitoring
28. Data from the daily monitoring of exposure conditions for
BRH sediment experiments one and two are presented in Table 1.
Table 1
Daily monitoring data for Black Rock Harbor
Experiments One and Two.*
Treatment
100 REF 50-50 BRH/REF 100 BRH
Experiment One
Actual
incoming 56.2 (8.2) 59.4 (5.5) 62.8 (9.9)
concentration
(mg particulates/fc)
Actual
surrounding 11.6 (5.1) 24.5(15.A) 30.2(17.5)
concentration
(mg particulates/£)
Experiment Two
Actual
incoming 49.4 (6.1) 52.9 (5.7) 56.2 (8.6)
concentration
(mg particulates/A)
Actual
surrounding 14.1 (6.4) 23.5(10.1) 29.0(11.4)
concentration
(mg particulates/£>
* Values are means with standard deviation in parentheses.
24
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29. The data presented in Table 1 indicate that comparable
levels of sediments were delivered to each treatment, and that those
levels were reasonably consistent throughout the course of the exper-
iments .
Physiological Parameters
30. The physiological results of the two experiments are
summarized in Tables 2 through 9. In the first experiment, two mussels
died during the SFG measurements, one from each of 100 REF and 50-50
BRH/REF treatments. As a result the mean values from these treatments
included only nine individuals, while the 100 BRH treatment means
represent ten mussels. No mortality occurred in the second experiment;
thus all mean values included ten individuals.
31. The weight-specific clearance rates are listed in Table 2.
In the first experiment, the 100 REF and 50-50 BRH/REF treatments were
similar while those mussels exposed to 100 BRH were significantly
lower. The results of the second BRH experiment indicate that the
mussels from the 100 REF treatment exhibited a significantly higher
clearance rate than the mussels from either of the other two treatments.
25
-------
Table 2
The mean (SE) weight-specific clearance rates of mussels
from the two BRH experiments.
Treatment Clearance Rate Group*
tt/hr)
100 REF
50-50 BRH/REF
100 BRH
100 REF
50-50 BRH/REF
100 BRH
Experiment One
3.81(0.23)
3.59(0.35)
2.25(0.44)
Experiment Two
3.99(0.37)
1.54(0.26)
1.40(0.34)
A
A
B
A
B
B
* Means with the same group letter are not significantly different.
32. The absorption efficiencies (Table 3) were all relatively
high in the first experiment with the 100 BRH and 100 REF treatments
significantly higher than the 50-50 BRH/REF group. In the second
experiment the absorption efficiencies were even higher; however, there
were no differences between any of the treatments.
Table 3
The mean (SE) absorption efficiencies of mussels
exposed to three exposure treatments in the two BRH experiments.
Treatment
100 BRH
100 REF
50-50 BRH/REF
100 REF
50-50 BRH/REF
100 BRH
Absorption Efficiency
(percent )
Experiment One
92(0.9)
89(0.8)
82(2.1)
Experiment Two
96(0.3)
96(0.3)
96(0.3)
Group*
A
A
B
A
A
A
* Means with the same group letter are not significantly different,
26
-------
33. The respiration rates for each treatment are listed in
Table 4. There were no significant differences between treatments
in either experiment. The actual rates were lower in the second
experiment than in the first possibly due to seasonal differences.
Table 4
Mean (SE) weight-specific respiration rates of
mussels exposed
Treatment
50-50 BRH/REF
100 REF
100 BRH
100 REF
100 BRH
50-50 BRH/REF
to three types of suspended
Respiration Rate
(ml-02/hr)
Experiment One
1.00(0.02)
0.91(0.04)
0.87(0.06)
Experiment Two
0.61(0.03)
0.53(0.04)
0.51(0.03)
sediments .
Group*
A
A
A
A
A
A
* Means with the same group letter are not significantly different.
34. The data listed in Table 5 indicate that the ammonia
excretion rates of the mussels from the 50-50 BRH/REF treatment were
significantly elevated as compared with those from the 100 REF group in
the first experiment, and the same differences were evident in the
second experiment.
27
-------
Table 5
The mean (SE) ammonia excretion rates for
mussels from the two BRH exposure experiments.
Treatment Ammonia Excretion Rate Group*
NH4-N/hr/g)
50-50 BRH/REF
100 BRH
100 REF
50-50 BRH/REF
100 BRH
100 REF
Experiment One
37.11(3.14)
26.88(2.81)
20.50(3.93)
Experiment Two
20.93(1.09)
19.42(2.48)
14.13(1.47)
A
A B
B
A
A B
B
* Means with the same group letter are not significantly different.
Scope for Growth Index
35. While the inter-experimental comparisons of the individual
physiological parameters are important, it is the comparison of the
integrative SFG index that is of prime interest. The weight-specific
SFG values for each treatment are listed in Table 6. In Experiment
One, the 100 REF group exhibited a significantly higher SFG than those
mussels exposed to 100 BRH and the 50-50 BRH/REF treatment. The same
relative treatment differences were observed in the second BRH exper-
iment, indicating the reproducibility of the technique. The actual
mean SFG value of the 100 REF treatment in the second experiment
(10.22 J/hr) was significantly higher than the first (2.53 J/hr),
possibly due to seasonal differences; however, the relative differences
were the same in both experiments. This will be further detailed in
the discussion section.
28
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Table 6
The mean (SE) weight-specific scope for growth values
for the three exposure treatments in the two
BRH experiments.
Treatment
100 REF
50-50 BRH/REF
100 BRH
100 REF
50-50 BRH/REF
100 BRH
Scope for Growth
(J/hr/g)
Experiment One
2.53(0.78)
-2.32(1.17)
-3.63(1.58)
Experiment Two
10.22(1.71)
0.51(2.01)
-1.07(1.80)
Group*
A
B
B
A
B
B
* Means with the same group letter are not significantly different.
36. Another index, the 0:N ratio, was calculated using the
respiration rate and ammonia-nitrogen excretion rate data. The
results, Table 7, indicate that there were no differences in these
values between treatments in the first experiment. In the second
experiment, the mean 0:N ratio of the 100 REF mussels was significantly
higher than 100 BRH mussels. The differences and inconsistencies
between the results of this index and the SFG index will be discussed
later in the report.
29
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Table 7
The mean 0;N ratios of each treatment in Experiments One and Two.
Treatment
100 REF
50-50 BRH/REF
100 BRH
100 REF
50-50 BRH/REF
100 BRH
0:N ratio
Experiment One
77
65
50
Experiment Two
63
49
34
Group*
A
A
A
A
A B
B
* Means with the same group letter are not significantly different.
37. One objective of the laboratory documentation phase of the
FVP is to investigate the variability of the SFG index. One way to do
this is to look at the coefficient of variation (CV) which is the
standard deviation divided by the mean. The absolute value of the CV
has been calculated for each physiological parameter and the SFG index
and is presented in Table 8.
30
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Table 8
The coefficients of variation (percent) for each of the
physiological parameters , the
Parameter
100
Experiment
Clearance rate
Absorption efficiency
Respiration rate
Ammonia excretion rate
Scope for growth
0:N ratio
Experiment
Clearance rate
Absorption efficiency
Respiration rate
Ammonia excretion rate
Scope for growth
0:N ratio
SFG
REF
One
18
3
14
58
92
51
Two
29
1
18
33
53
36
index, and the 0:N
Treatment
50-50 BRH/REF
29
8
7
25
152
43
53
1
18
16
1250
22
ratio.
100 BRH
63
3
21
33
138
25
76
1
24
40
530
47
38. The data in Table 8 would indicate that exposure to BRH
material causes a great increase in the variability associated with the
clearance rate measurement and the SFG index in both experiments.
39. The means of the dry weights and lengths of the mussels from
each treatment are listed in Table 9. The results from Experiment One
indicate that the lengths of the mussels from each treatment were not
significantly different, with coefficients of variation, CV, of 2-3
percent. The dry weights of those same animals indicated that the
mussels from the 100 REF treatment were significantly higher than the
other two treatments, with the CV increasing directly with increased
exposure to BRH material. The results of the second experiment also
indicated that the lengths of the mussels used were very similar.
31
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Unlike the first experiment, however, there were no differences between
the dry weights of the animals from each treatment. There was no
evidence of spawning in any of the treatments.
Table 9
Mean dry weight and length of mussels
from each treatment in the
BRH experiments.
Treatment
Dry Weight
(g)
Group* CV**
f tjf V
\/o J
Experiment
100
50-50
100
REF
BRH/REF
BRH
0
0
0
.86(0
.59(0
.59(0
.04)
.04)
.06)
A
B
B
Experiment
100
50-50
100
REF
BRH/REF
BRH
0
0
0
.91(0
.88(0
.73(0
.05)
.04)
.05)
A
A
A
One
15
21
32
Two
24
15
24
5
5
5
5
5
5
Length
(cm)
.32(0.
.32(0.
.41(0.
.45(0.
.46(0.
.40(0.
05)
04)
05)
04)
01)
01)
Group*
A
A
A
A
A
A
CV
3
2
3
4
2
2
* Means with the same group letter are not significantly different,
** CV - coefficient of variation.
32
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PART IV: DISCUSSION
40. The objective of the studies reported here is to evaluate
the sensitivity, variability, and reproducibility of the SFG index as
a method to assess the effects of BRH dredged material on M. edulis
in a laboratory exposure. As shown in Table 6, a significant reduction
in the SFG index was observed in mussels exposed to BRH dredged material
when compared to mussels exposed to REF sediment, thus demonstrating
the sensitivity of this index.
41. The BRH dredged material contains polychlorinated biphenyls
(PCB) (6800 ng/g), polynuclear hydrocarbons (PAH) (9800 ng/g), and
trace metals (Cu, Cr, and Pb, at 2380, 1430, and 380 ug/g, respectively)
(Lake et al. 1984). In the same paper M. edulis was reported to
accumulate 44% of the sediment PCB's, 28% of the sediment polynuclear
hydrocarbons, and various amounts of the Cu, Cr, and Pb trace metals
during a 28-day exposure to the BRH material. It is likely that
the same materials were accumulated by the mussels during this experi-
ment. A correlation is indicated between the significant differences
in the physiological parameters and SFG values and the contaminants
in the BRH material.
42. The data in Experiment One indicated that the mussels from
the 100 BRH treatment exhibited1 a significantly lower clearance rate
when compared to the other two treatments (Table 2). Stickle et al.
(1983) reported a decreased clearance rate in M^ edulis after a 28-
day exposure to the water-soluble fraction of crude oil. Abel (1976)
33
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investigated the effects of several metals on filtration rates in
Mytilus and found that Cu, Zn, and Hg caused a reduction in this
physiological parameter. Gilfillan et al. (1976) reported a reduced
carbon flux in the soft-shelled clam, Mya arenaria, from areas that
had been exposed to an oil spill. They attributed this to decreased
filtration rates and higher respiration rates. Gilfillan (1975)
showed that exposure to crude oil extracts also caused a reduction
in filtration rates in M. edulis. Thus, sufficient evidence exists
to support the hypothesis that individual exposures to heavy metals
or oils can cause reduced filtration rates.
43. The results of the second experiment were similar with
differences again between the clearance rates in the 100 REF and 100
BRH treatments. However, in the second experiment the mussels from the
50-50 BRH/REF treatment also exhibited a significantly reduced clear-
ance rate from those mussels in the 100 REF treatment. While the
reason for this is not immediately obvious, one possible explanation
may be forwarded. The concentration of BRH material in the 50-50
BRH/REF treatment, 25 mg/£ of BRH material, may be near the threshold
level that produces this effect. If the mussels filtered slightly
more in the second experiment than the first, their exposure to the
the BRH material would have been increased. A significant decrease
in the clearance rates of the 100 BRH mussels was observed in both
experiments. Therefore, a slight difference in exposure level
(i.e., clearance rate) in the 50-50 BRH/REF treatment may have pro-
duced the reduced clearance rate in the second experiment.
34
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44. In addition to affecting the absolute clearance rate, BRH
dredged material also resulted in an increase in the variability of
the clearance rate measurement (Table 8). In both experiments the
response was consistent: an increased exposure to BRH material
resulted in a corresponding increase in the CV of this measurement.
45. In contrast to the lower clearance rates, no differences
were observed in respiration rates between the treatments in either
BRH experiment. Stickle et al. (1983) found no differences in respira-
tion rates between treatments in the exposures to crude oil previously
mentioned. During acute tests, Brown and Newell (1972) observed a
decrease in whole animal respiration rates in M^ edulis during expo-
sure to Cu. Scott and Major (1972) reported a similar decrease in
the same species. In contrast, Gilfillan et al. (1976) stated that
reduced carbon flow in M. arenaria was partially due to Increased
respiration rates. Engel and Fowler (1979) found that Cu caused an
increase in respiration rate in excised oyster gill tissue. It is
evident from the literature that no one respiration rate response is
elicited consistently after exposure to a pollutant. While no
differences in respiration rates were evident in the present study,
it is important, for the purposes of this report, to note that the
same response was obtained in both experiments.
46. The absorption efficiencies in both experiments were quite
high. In the first experiment the 50-50 BRH/REF treatment was signif-
icantly lower than the 100 REF and 100 BRH treatments (Table 3),
while in the second experiment there were no differences between any
treatment. The algae used in this study were T. Iso, a naked flagellate.
-------
This fact, along with the low food concentrations, 0.5 mg/S, used
during the SFG measurements, can account for these high efficiencies.
Widdows, Phelps, and Galloway (1981) fed Tetraselrois suecica to mussels
at a concentration of 0.35 rng/d and found similarly high absorption
efficiencies of 93 percent. Other reported absorption efficiencies
that use such species as the diatom Phaeodactylum tricornutum or
natural seston would be expected to be lower than those reported
here.
47. While each individual physiological parameter measured is of
interest, the advantage of the SFG index is that it integrates the
whole animal response of each individual. In the first experiment, the
SFG data indicate that the mussels exposed to the 50-50 BRH/REF and 100
BRH treatments exhibited significantly lower SFG values, -2.32 and
-3.63 J/hr, respectively, than those mussels from the 100 REF treatment
(2.53 J/hr). The results of the second BRH experiment were the same as
the first one, demonstrating the reproducibility of the technique.
Those mussels from the 100 REF treatment displayed a significantly
higher SFG (10.22 J/hr) than mussels from the 50-50 BRH/REF (0.51 J/hr)
or 100 BRH (-1.07 J/hr) treatments. The SFG results are typical of a
dose-response effect. A similar decrease in SFG was reported for M._
edulls by Stickle et al. (1983) and for M^ arenarla by Gilfillan et al.
(1976) following exposure to oil. Widdows et al. (1981) have also
reported a dose response type effect between SFG and aromatic petroleum
hydrocarbon exposure concentrations in M. edulls.
36
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48. The results of another index, 0:N ratio, are listed in
Table 7. With this index, a lower 0:N ratio is indicative of a more
stressed condition. In Experiment One there were no differences
between treatments using this index. The results of the second
experiment, however, indicated that the 100 REF treatment mussels
exhibited a significantly higher 0:N ratio than those mussels from
the 100 BRH treatment. The results of this index are inconsistent
with respect to the SFG index and they are also different between
the two experiments. In addition, the 0:N ratio results do not
follow the dry weight data listed in Table 9. The dry weight data
from Experiment One indicated that the 100 REF treatment mussels
weighed significantly more than the mussels from the other two treat-
ments. This is the same pattern that the SFG index showed. In the
second experiment, there were no significant differences between
treatments; however, the order was the same as in the first experiment,
as were the SFG results. The dry weight measurements listed in
Table 9 were made initially to calculate weight-specific physiological
rates, not to infer changes due to treatment differences. One may
infer, however, that an index used to measure stress, such as SFG
and 0:N ratio, might parallel changes in actual tissue weight. In
these experiments the SFG index corresponds more closely to these
changes than do the 0:N ratio.
37
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PART V: CONCLUSIONS AND RECOMMENDATIONS
49. The major objectives of the laboratory documentation portion
of the Field Verification Program were to assess the sensitivity,
reproducibility, and variability associated with various biological
indices using BRH dredged material. The results of this experiment
indicate that the Scope For Growth index, with M^ edulis as the test
organism, is a sensitive enough technique for measuring the chronic
effects of this dredged material.
50. The primary purpose of this report, however, was to docu-
ment the reproducibility of this index. The data in Table 6 indicate
that the results of one experiment can be reproduced in a second
replicate study. The SFG value of the 100 REF treatment from experi-
ment two (10.22 J/hr) was significantly higher than the 100 REF SFG
value from the first experiment (2.53 J/hr). This is not unexpected
because SFG values do change normally during the year with changes
in the reproductive cycle (Bayne et al. 1976). The important point
is that the SFG values indicated the same relative response in both
BRH experiments. This was the hypothesis being tested in this exper-
iment and the rationale for using standardized conditions in the SFG
measurements. The results of this experiment indicate that the SFG
index, when used with M. edulis, satisfies the question of repro-
ducibility for the Laboratory Documentation phase of the FVP.
51. In addition to measuring the sensitivity and reproduc-
ibility of the SFG index, the effect this material has on the varia-
bility of the measurements was also of interest. The data presented in
38
-------
Table 8 would indicate that this material increases the variability
observed in several of the measurements. The effect of BRH material
on the variability of the clearance rates has already been mentioned.
Clearance rate differences have a dramatic effect on the total amount
of energy consumed, and, therefore, on the subsequent calculation of
the SFG index. This can also be seen from Table 8, where large
variation was also observed in the SFG index. The exact nature of
how BRH causes more variability in this measurement is not clear.
However, it does point out one advantage of using an index such as
SFG; the physiological system that is negatively impacted may be
isolated. In these two experiments it was apparent that BRH material
had an effect on clearance rate. The actual mechanistic basis for
this effect can now be studied further.
39
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Mytilus," Marine Pollution Bulletin, vol 7, pp 228-231.
American Society for Testing and Materials, 1980. "Standard Practice for
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Amphibians," ASTM E-729-80, Philadelphia, Pa.
Bayne, B.L. 1976. "Aspects of Reproduction in Bivalve Molluscs,"
Estuarine Processes. Vol. 1; Uses, Stresses and Adaption to the
Estuary, Edited by Wiley. Academic Press, London, pp 432-448.
Bayne, B.L., Clark, K.R., and Moore, M.N. 1981. "Some Practical
Considerations in the Measurement of Pollution Effects on Bivalve
Molluscs, and Some Possible Ecological Consequences," Aquatic
Toxicology, vol 1, pp 159-174.
Bayne, B.L., Moore, M.N., Widdows, J., Livingstone, D.R., and Salkeld,
P.N. 1979. "Measurement of the Responses of Individuals to
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integrations," Marine Mussels: Their Ecology and Physiology, Edited by
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Bower, C.E., and Holm-Hansen, T. 1980. "A Salicylate-Hypochlorate
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Brown, B.E., and Newell, R.C. 1972. "The Effect of Copper and Zinc on
the Metabolism of the Mussel Mytilus edulis," Marine Biology, vol 16, pp
108-118.
Conover, R.J. 1966. "Assimilation of organic matter by zooplankton,"
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Consumption in Animal Energetics," Oecologia, vol 19, pp 195-201.
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Gilfillan, E.S. 1975. "Decrease of Net Carbon Flux in Two Species of
Mussels Caused by Extracts of Crude Oil," Marine Biology, vol 29, pp
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Gilfillan, E.S., Mayo, D., Hanson, S., Donovan, D., and Jiang, L.C.
1976. "Reduction in Carbon Flux in Mya arenaria Caused by a Spill of
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Contaminants from Black Rock Harbor Dredged Material by Mussels and
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of mussels from Narragansett Bay."
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mussels from Narragansett Bay."
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State University Press, Iowa, 593 pp.
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"Bioenergetics and Survival of the Marine Mussel, Mytilus edulis,
During Long-term Exposure to the Water Soluble Fraction of Cook Inlet
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Thompson, R.J., and Bayne, B.L. 1974. "Some Relationships Between
Growth, Metabolism and Food in the Mussel, Mytilus edulis,'' Marine
Biology, vol 27, pp 317-328.
. 1972. "Active Metabolism Associated with Feeding in
the Mussel Mytilus edulis," Journal of Experimental Marine Biology and
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Feeding, Bioenergetics and Growth of Fish," The Biological Basis of
Freshwater Fish Production, Edited by Gerking, S.D., Blackwell
Scientific Publications, Oxford, pp 174-219.
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Moore, M.N., and Salkeld, P.N. 1981. "Measurement of the Responses of
Mussels to Environmental Stress and Pollutants in Sullom Voe: A
Baseline Study," Proceedings of the Royal Society of Edinbergh, vol 80
B, pp 323-338.
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Between Season, Available Food and Feeding Activity in the Common
Mussel Mytilus edulis," Marine Biology, vol 50, pp 195-207.
Widdows, J., Phelps, D.K., and Galloway, W.B. 1981. "Measurement of
Physiological Condition of Mussels Transplanted Along a Pollution
Gradient in Narragansett Bay," Marine Environmental Research, vol 4, pp
181-194.
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Aquaculture Systems," Aquaculture, vol 13, pp 1-33.
42
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APPENDIX A
NO-OBSERVABLE-EFFECT-CONCENTBATION EXPERIMENT
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Introduction
1. As stated in the main text it was determined that in order
to obtain an effect with BRH material it was believed that the mussels
should be exposed to the highest reasonable level of suspended partic-
ulates. The determination of a reasonable particulate level was
termed the no-observable-effeet-concentration (NOEC) experiment.
Two criteria were used in selecting the NOEC: (a) maximum exposure
to the suspended sediment, and (b) no significant reduction in SFG.
The null hypothesis was that there was no particulate level of sus-
pended reference sediment that would have a chronic effect on the
mussels. As described in Part I of the main text, SFG was measured
under standardized conditions to determine whether some chronic
effect had been incurred during the 28-day exposure.
Material and Methods
2. To test this hypothesis, reference sediment (REF) from
Long Island Sound (Lake et al. 1984) was used and mussels were exposed
in groups of twelve each to the following nominal dilution series:
100, 50, 25, 12.5, 6.25, and 0 mg/£. Figure 4 shows the mussel
exposure system. Sediment was delivered to the mixing chamber from
the composite dosing system through a microprocessor-controlled
valve as previously reported by Lake et al. (1984). For this experi-
ment, the mixing and distribution chambers labeled BRH in Figure 4
contained filtered seawater only, while total suspended particulates
(100 mg/£) were maintained in the REF mixing and distribution
chambers. The various dilutions were achieved by blending suspended
A2
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particulates from the REF distribution chamber with the filtered sea-
water. Each exposure chamber contained 12 mussels and received the
appropriate mixture at a rate of 100 ml/min. The duration of the
experiment was 28 days with samples of mussels taken on day 28 for
SFG determinations. The procedures used for measuring SFG were the
same as those descibed in the main text. The highest particulate
level that did not produce a negative lasting effect would be chosen
as the NOEC for the two BRH exposure experiments.
3. Measurements of the amount of suspended particulates
entering the exposure chambers were made daily using a spectrophotom-
eter, as previously detailed in Part II. The actual incoming and
surrounding suspended particulate concentrations are shown in Table Al,
Table Al
Results of daily monitoring of exposure system
in the NOEC experiment.
Treatment
Control 6.25 12.5 25 50 100
Actual
incoming
cone.
(mg/A)
Actual
surrounding
concentration
2(0.5) 7(2.3) 12(4.5) 26(6.2) 53(13.8) 106(27.6)
7(8.0) 10(7.1) 12(6.5) 15(6.9) 21(9.3) 49(15.6)
Algae 0.094 0.094 0.094 0.094 0.094
(g/mussel/day)
Ref sediment
(g/mussel/day)
Percent Food
0.0 0.075 0.150 0.300 0.600
100 55.6 38.5 23.9 13.5
0.094
1.200
7.3
A3
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4. The mussels were continuously fed laboratory cultured
T-Iso at a rate of 94 mg (dry weight) per mussel per day (Table Al).
Conditions and techniques of algal culture were modified after Guillard
(1975). Guillard's "f/2" nutrient media was used, except that all
trace metals but iron were eliminated and the concentration of the
vitamins thiamine and B12 were doubled.
Results
Physiological parameters
5. Results of the physiological measurements are summarized
in Tables A2 through A5. They indicate no consistent pattern between
each parameter and the final SFG values (Table A6). The most probable
reason for this is that some mussels from all treatments, except
100 mg/i, were observed spawning during the physiological measurements
(Table A7). This point will be discussed more fully in the following
Discussion Section but should be kept in mind as the results in
Tables A2 through A5 are presented.
6. The results of the reference sediment experiment are sum-
marized in Tables A2 through A7. Table A2 lists the weight-specific
mean clearance rates for the mussels from the six treatment levels.
Although there was a wide range in the mean values, there were no
significant differences between any of the treatments.
A4
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Table A2
Comparison of mean (standard error) weight-specific
clearance r
Treatment
100
50
Control
12
25
6
ates of ten mussels pei
test with reference
Clearance Rate
(jj/hr)
4.22(0.45)
4.13(0.42)
3.43(0.40)
2.95(0.52)
2.93(0.19)
2.64(0.39)
: treatment in the NOEC
sediment.
Group*
A
A
A
A
A
A
* Means with the same group letter are not significantly different.
7. The absorption efficiency results are listed in Table A3.
Again there is a large variation in the means. The mussels from the 25-
mg/» treatment were significantly lower than the mussels from the 50-,
control, and 100-mg/Jl treatments.
Table A3
The mean (SE) absorption efficiencies of ten mussels per
treatment
Treatment
•g/l
50
CONTROL
100
12
6
25
in the NOEC reference sediment
Absorption Efficiency
(percent)
76(0.04)
73(0.03)
72(0.03)
66(0.05)
56(0.06)
51(0.07)
experiment .
Group*
A
A
A
A B
A B
B
* Means with the same group letter are not significantly different.
8. The mean weight-specific respiration rate for each treatment
is listed in Table A4. The control group was significantly higher than
all other groups.
A5
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Table A4
The mean (SE) weight-specific respiration rates for
Treatment
mg/ft
Control
25
100
50
6
12
the NOEC experiment.
Respiration Rate
(ml 02/hr/g)
1.20(0.07)
0.95(0.05)
0.95(0.05)
0.84(0.06)
0.79(0.03)
0.78(0.02)
Group*
A
B
B
B
B
B
* Means with the same group letter are not significantly different.
9. Ammonia excretion rate means for each treatment are listed
in Table A5. The mussels from the 6-mg/£ treatment had a significantly
higher mean excretion rate than those mussels from the 12-mg/& and
control treatments. No other differences were observed.
Table A5
Mean (SE) ammonia excretion rates for ten
mussels from each
Treatment
mg/A
6
25
100
50
12
Control
treatment from the
Excretion Rate
(yg NH4-N/hr/g)
29.07(4.64)
21.48(4.59)
20.33(5.91)
15.05(3.12)
11.80(2.19)
7.99(1.99)
NOEC experiment.
Group*
A
A B
A B
A B
B
B
* Means with the same group letter are not significantly different.
Scope for growth index
10. The mean SFG values for each treatment in the NOEC experiment
are listed in Table A6. The 50-, 100-, and 12-mg/A treatments were
statistically similar as were the 12-, 6-, control, and 25-mg/A groups.
A6
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In addition, the 100-, 12-, and 6-mg/Jl treatments were not statis-
tically different. There was a large amount of variability in this
data set which is reflected in the 95% confidence limits.
Table A6
The mean(SE) weight-specific scope for growth values
Also
Treatment
mg/A
50
100
12
6
Control
25
for each treatment
listed are the 95% <
Scope For Growth
(Joules/hr/g)
1.73(1.85)
-0.96(1.13)
-3.86(1.42)
-7.07(0.98)
-8.48(1.48)
-9.40(1.58)
in the NOEC ex
confidence limi
Group*
A
A B
ABC
B C
C
C
periment.
ts for each mean.
95% Confidence Limits
5.85 to -2.29
1.56 to -3.48
-0.70 to -7.02
-4.89 to -9.25
-6.30 to -10.66
-5.88 to -12.92
* Means with the same group letter are not significantly different.
11. Several other measurements were completed on these mussels
and are listed in Table A7. There was no difference between the mean
length of the mussels in each treatment; however, the dry weight of
the mussels from the 12- and 50-mg/£ treatments were statistically
higher than those from the 100-mg/fc group. In addition, several
mussels were observed spawning during and/or after the clearance rate
measurements. While these numbers are listed in Table A7, there is no
assurance that these were the only mussels to have spawned. Other
mussels may have spawned either at night or prior to any of the
physiological measurements.
A7
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Table A7
Mean (SE) lengths and dry weights of ten mussels from
each treatment in the NOEC experiment. Also listed are the
number of mussels observed spawning during physiological
measurements from each treatment.
Treatment Length(SE) * Dry Weight(SE)* Mussels Spawning
mg/A (cm) (g)
12
50
25
6
Control
100
5.33(0.06) A
5.40(0.06) A
5.35(0.04) A
5.33(0.05) A
5.41(0.04) A
5.35(0.05) A
0.66(0.07) A
0.65(0.04) A
0.62(0.03) A B
0.60(0.03) A B
0.55(0.02) A B
0.47(0.04) B
1
1
2
1
8
0
Means with the same group letter are not significantly different.
A8
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Discussion
12. The purpose this preliminary experiment was to determine
a suspended sediment load that would have no observeable effect on
the SFG of Mytilus edulis. This concentration was determined to be
a nominal incoming concentration of 50 mg/&, based on two facts:
(a) psuedofeces were continuously produced in this treatment, thus
giving maximum exposure to the suspended sediment; and (b) the mean
SFG value was highest for this treatment. This decision may seem
arbitrary after inspection of the results of this experiment. A
great deal of variability and inconsistency occurred in the data
from this experiment, most probably associated with the spawning
activity during the measurements. However, for the purpose of this
report, which is to test the accuracy and reproducibility of the SFG
index, the results are quite important and are included as this appendix.
13. The most obvious result of this experiment was the lack
of a consistent pattern in the response of the individual physiological
parameters used to calculate the SFG index, relative to the exposure
level. SFG is an integrative index; however, the data from individual
physiological measures can be important to the interpretation of
results. In previous studies the effects of particular pollutants
have been documented with M. edulis (Phelps et al. 1983, Stickle
et al. 1983, Widdows at al. 1981, Bayne et al. 1979). In these
studies one of the measured SFG parameters contributed overwhelmingly
to the observed change in SFG. The reference sediment used in the
present experiment was from the relatively clean reference site in
A9
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Long Island Sound. The effects observed in the present NOEC experi-
ment, therefore, are probably due to physical action of the suspended
material rather than to a toxic chemical compound. The results of
the NOEC experiment showed no clear pattern between the individual
physiological paramerers in response to the sediment load. Three
possible explanations are considered. First, the exposure system
itself may have introduced variability due to some unidentified
artifact. Second, the physiological measurements may have introduced ,
some unknown error into the data set. Third, a factor other than
sediment concentration, i.e., differences in the reproductive condition
of mussels between treatments, may have been influencing the results.
14. After inspection of the system monitoring data, it would
appear that the first possibility is not likely. Table Al shows that
the system was working properly throughout the experiment.
15. The second alternative would appear to be unsubstantiated
as well. For example, the clearance rate data indicated that the
mussels with the highest suspended levels during the experiment (50
and 100 mg/jj,) also had the highest clearance rates (Table A2)
during the SFG measurements. If this were an artifact from the
experiment, one would expect just the opposite. Winter (1978) stated
that mussels try to maintain a constant ingestion rate by decreasing
clearance rate with increasing particle concentration. This same
effect was described by Widdows et al. (1979). Therefore, the observed
clearance rates do not seem to be due to some factor such as improper
acclimation time before the clearance rate measurement was initiated.
A10
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16. The absorption efficiency data likewise follow no distinct
pattern. While variable, the only significant differences were between
the 50- and 25-mg/£ treatments (Table A3). Thompson and Bayne (1972)
showed that absorption efficiency was inversely proportional to the
suspended particulate load. If something from the sediment experiment
were affecting this measurement, one would expect differences between
the highest and lowest particulate concentrations. In addition, the
respiration rates were highest in the control and 100-mg/^ treatments,
which had the greatest difference in particle levels during the test.
17. The important conclusion is that no individual physiological
factor was apparently responsible for the SFG differences between
treatments. In this experiment the factor most closely correlated to
the order of SFG values was the total suspended particulate levels,
and consequently food levels. The amount of food supplied to each
treatment was the same; however, the particulate levels were different
(Table Al). In effect, the reference sediment caused a decrease in
the percentage of algae ingested with increasing particle concentration
(Table Al). This had the effect of supplying more algae to the control
group and proportionally less to each higher particulate level group.
18. Differences in food levels between treatments lead to the
third alternative, which offers the most probable explanation for the
observed variability and inconsistencies in the NOEC experiment.
Sastry (1975), working with another bivalve, Argopectin irradians, found
that both an adequate food supply and temperature regime were necessary
to initiate the gametogenic cycle in the bay scallop. Bayne (1976)
All
-------
postulated that the same process was necessary with M. edulis. In
the present experiment, it is possible that the combination of temper-
ature (constant 15°C) and adequate food supply in the control and
lower particle level treatments may have been sufficient to initiate
the gametogenic cycle. The higher concentrations may not have had a
sufficient food supply for gametogenesis to occur at the same rate as
the lower concentrations. The number of mussels spawning during the
SFG measurements may add some credence to this assumption (Table A7).
The control treatment had eight out of ten mussels at least partially
spawn, while the 25-mg/fc treatment had two spawn, and one mussel out
of ten in each of the 6-, 12-, and 50-mg/S- treatments. No spawning
occurred in the 100-mg/i, treatment mussels. Bayne et al. (1976) stated
that SFG values are lower during gametogenesis, and this may be a
possible explanation for the variable results. While this hypothesis is
speculative at the present time, inspection of the reproductive cycle
of the field population from which these mussels were collected may
help to clarify it.
19. In another study, Nelson (in prep) followed the gameto-
genic cycle of this population during the past year. , Using sterology
and mantle dry weight as reproductive indices, he found that mussels
from this area exhibited a large spawning peak in late March and a
smaller peak in early December. This second spawning period coincides
with the collection period for the NOEC experiment. The mussels
used in this experiment, therefore, were probably at different stages
of the gametogenic cycle. This fact, in addition to the possible
A12
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treatment food differences mentioned above, may help to explain the
spawning differences observed in this experiment. The important
point is that differences in the reproductive condition of experimental
organisms can lead to serious problems when the data are interpreted.
This is one factor that must be considered in any experimental design.
20. With this problem in mind, the SFG values from this experi-
ment will be discussed briefly. These values can only be compared
in a relative sense because the physiological measurements were
completed under standardized conditions. A mussel with an SFG value
of zero is, by definition, at its maintenance ration. Inspection of
the 95 percent confidence limits about the means indicate that the
50-, 100-, and 12-mg/Jl treatments were around that maintenance ration.
The other three treatments were below this ration level for this test
only. During the physiological measurements, cultured algae were
supplied at approximately 0.5 mg/£ of seawater. This ration was
chosen based on the metabolic rates of mussels collected at this
time of the year in the field (Nelson in prep). Because this
test was completed in less than 24 hr, it is believed that any sub-
maintenance ration did not adversely affect the animals. In a rela-
tive sense, therefore, it is believed that the statistical differences
observed between means are valid. Long-term holding under these
conditions would not be recommended. However, in the exposure system,
the food levels were considerably higher.
21. The final SFG values (Table A6) indicate that the mussels in
the 50-, 100-, and 12-mg/fc treatments were not different statistically.
A13
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Of these three, only the 50-mg/A treatment did not overlap any other
group. In light of this fact and the previous information concerning
pseudofecal production, a nominal incoming concentration of 50 mg/& was
chosen as the NOEC for the BRH experiments.
22. One problem encountered during the NOEC test was the repro-
ductive condition of different groups of mussels. This problem was
not encountered during the BRH experiment. Differences in reproductive
condition can cause complications when interpreting SFG data measured
at different times of the year. One possible solution would be to
use juvenile mussels in place of adults. The use of adult mussels
to measure the effects of pollutants, including SFG, is well documented.
With the proper experimental design a hypothesis could be tested to
determine if the same results could be achieved using juvenile mussels
instead of adults. This would alleviate one source of variability
and possibly make the comparison of SFG results more straightforward.
A14
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